Condiciones particulares de los elementos de ventilación 1. Aberturas y bocas de ventilación

Figure 4-5. IRF4^f/f CD11c-cre-positive bone marrow chimeras manifest no changes in overall tissue cell numbers but have fewer dendritic cells

Total number of cells collected from digested small intestines (SI), colons, sMLNs and cMLNs of CD11c-cre-positive or IRF4^f/f cre-negative bone marrow chimeras and assessed the total number of cells and total DCs in these tissues. Small intestines, colons and MLNs were enzymatically digested and after cell straining single cell suspensions were counted and total cell numbers recorded for each tissue. As expected around 20 million cells could be isolated from the small intestine and around 7 million cells were obtained from the colon. We collected 12 million cells from sMLNs and 4 million from cMLNs. No differences in total cell numbers were observed between cre+ and cre- mice in these tissues (Fig. 4-5 A), indicating that the deletion of IRF4 in CD11c-expressing cells did not cause major disruption in the tissues, such as tissue damage or developmental defects.

We stained single cell suspensions from each tissue with fluorescent monoclonal antibodies and were able to distinguish DC populations as previously described. In the intestines total DCs were selected as live single CD45⁺ B220^- MHCII⁺ CD11c⁺ CD64^- Ly6C^- cells and in the MLNs migratory DCs were distinguished by gating on live single B220^- MHCII^hi CD11c⁺ Ly6C^- cells. DCs accounted for around 0.5-1% of

all total cells. We had previously shown that DCs where the major IRF4-expressing CD11c⁺ cell population in the intestine and observed a general decrease of DCs in the intestines and MLNs of IRF4^f/f CD11c-cre-positive bone marrow chimeras. Despite not being statistically significant, the percentage of DCs tended to decrease in the small intestine and decreased to a significant level in the colon. Significant decreases were also observed in the percentage of migratory DCs in the sMLNs and cMLNs (Fig. 4-5 B). Using the total number of cells that we had counted and the respective percentage of DCs we calculated the number of DCs for each sample. Again we observed a reduction in the numbers of DCs in all tissues. Here numbers decreased significantly in the small intestine, colon and cMLN (Fig. 4-5 C).

We concluded that the deletion of IRF4 in DCs did not have a general impact on the integrity of the intestines or MLNs, as total cell numbers were comparable between IRF4^f/f CD11c-cre-positive and IRF4^f/f cre-negative bone marrow chimeras. However, DCs were reduced both in percentage and total number after IRF4 deletion, which suggested that IRF4 had an impact on DC development or differentiation.

Figure 4-6. IRF4 deletion in CD11c⁺ cells leads to a decrease in DP DCs in the small intestine and to a general reduction of DCs in the colon

DC subsets were identified by flow cytometry in small intestinal cell suspensions of IRF4^f/f CD11c-cre-positive and IRF4^f/f cre-negative bone marrow chimeras (A). The percentage (B) and total

To assess whether the impact of IRF4 on DC development was subset specific or if it affected all DCs equally we analysed small intestinal and colonic LP DC subsets of IRF4^f/f CD11c-cre-positive and IRF4^f/f cre-negative bone marrow chimeras. It has previously been reported that the deletion of IRF4 resulted in a decrease of the DP DC subset in the small intestine (Persson et al., 2013b). After digesting the small intestine with RPMI containing 1 mg/ml Collagenase VIII and 10% FCS, single cell suspensions were obtained by cell straining and stained for flow cytometry. DCs were selected as live single CD45⁺ B220^- MHCII⁺ CD11c⁺ CD64^- Ly6C^- cells and separated into four subsets by the expression of CD11b and CD103. We observed that in IRF4^f/f CD11c-cre-positive bone marrow chimeras the distribution of DC subsets was proportionally different compared to IRF4^f/f cre-negative controls. We observed that the proportion of DP DCs decreased from 35% in cre- mice to around 20% in cre+ mice. Furthermore, we observed that CD11b⁺ and CD103⁺ subsets increased proportionally (Fig. 4-6 A&B). To test whether this increase of CD11b⁺ and CD103⁺ DCs was caused by

CD1

an increase in cell numbers, or was just a proportional increase to compensate for the decrease in DP DCs, we analysed total cell numbers. Hereby total tissue cell counts were multiplied with the percentage of total DCs and the percentage of each respective subset. We observed that the number of DP DCs decreased significantly by the deletion of IRF4. The absolute numbers of CD11b⁺ and CD103⁺ DCs however were not affected (Fig. 4-6 C). We concluded that the DP DC subset was the only subset in the small intestinal lamina propria that was affected by the deletion of IRF4 and decreased in number, confirming already published findings (Persson et al., 2013b).

However, it remained unknown how the deletion of IRF4 in CD11c cells affected the composition of DC subsets in the colonic lamina propria. We harvested the colons of IRF4^f/f CD11c-cre-positive and IRF4^f/f cre-negative bone marrow chimeric mice and digested them enzymatically. Cells were stained with fluorescent monoclonal antibodies and DC subsets identified as CD11b/CD103 expressing live single CD45⁺ B220^- MHCII⁺ CD11c⁺ CD64^- Ly6C^- cells. Whereas DP and CD103⁺ DCs comprised the majority of DCs in the small intestine, CD11b⁺ and CD103⁺ DCs were the most abundant subsets in the colon. We observed that the proportion of DC subsets was different in cre+ mice, with an increase of CD11b⁺ DCs from 30% to 40% and a reciprocal decrease of DP DCs from 15% to 5% (Fig. 4-6 D&E). The proportion of CD103⁺ and DN DCs was not statistically affected but CD103⁺ were slightly increased. These proportional changes were similar to our observations in the small intestine and showed a decrease of DP DCs and a reciprocal increase of CD11b⁺ and CD103⁺ DCs.

However, the analysis of total cell numbers revealed that only DP DCs were decreased in the small intestine. When we analysed total cell numbers of colonic DCs we observed that they were generally 10-fold lower compared to the small intestine, due to a lower percentage of DCs and smaller tissue size. This difference had been observed in C57BL/6, cre+ and cre- mice and has also been reported in the literature (Denning et al., 2011; Houston et al., 2016). Comparing total cell numbers between IRF4^f/f CD11c-cre-positive and IRF4^f/f cre-negative mice in the colon, we noticed that all DC subsets were reduced. Hereby, DP DCs were the most affected population, whereas CD103⁺ DCs showed the least decrease in cell numbers (Fig. 4-6 F). This suggested that IRF4 deletion had a

lamina propria. Overall, DP DCs were the most affected subset in both the small intestine and colon, whereas CD103⁺ DCs were the least affected. We therefore hypothesized that DP DCs were important for the induction of Th2 responses and that their decrease was the cause for the defective Th2 response in IRF4^f/f CD11c-cre-positive bone marrow chimeras after egg injection.

Figure 4-7. Antigen uptake by intestinal DCs is not affected by the deletion of IRF4

20 µg of AlexaFluor660-labled SEA was injected into the small intestinal lamina propria of IRF4^f/f CD11c-cre-positive and IRF4^f/f cre-negative bone marrow chimeras. Levels of SEA uptake by DCs were measured 24 hours after injection (A). Total numbers of AF660-SEA⁺ SI LP DCs were compared between cre+ and cre- mice (B). SEA-AF660 was also injected into the colonic lamina propria and uptake by LP DCs assessed after 24 hours (C). Total numbers of AF660-SEA⁺ colon LP DCs were compared between cre+ and cre- mice (D). Data represent at least three independent experiments (mean ± SEM) with each point representing one animal. Mann-Whitney U tests were applied between cre+ and cre- groups for statistical analysis.

As we hypothesised that the decrease of DP DCs in the small intestinal lamina propria and the colon affected the induction of Th2 responses we tested whether antigen uptake by lamina propria DCs was adversely affected in IRF4^f/f CD11c-cre-positive mice. We fluorescently labelled soluble egg antigen with AlexaFluor660 and injected 20 µg into the small intestinal lamina propria of IRF4^f/f CD11c-cre-positive and IRF4^f/f cre-negative bone marrow chimeric mice. From previous experiments we knew that this amount was equivalent to amounts used to drive robust Th2 responses, which suggested that it was likely to be taken up by DCs in the lamina propria and transported to the draining lymph nodes. 24 hours

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after AF660-SEA injection the injected area of the small intestine was excised and digested with Collagenase VIII. Cells were stained with fluorescent monoclonal antibodies and analysed by flow cytometry. We gated on total lamina propria DCs and analysed the level of AF660 fluorescence as a measure of antigen uptake. We observed that around 8% of all DCs labelled positive for AF660-SEA in both cre+

and cre- mice (Fig. 4-7 A). This suggested that despite the change in DC subset composition in cre+ mice uptake of parasite antigen was not affected in the lamina propria. This was confirmed by comparing the total number of SEA-labelled DCs in the small intestine where also no difference between cre+ and cre- mice was observed (Fig. 4-7 B).

We had observed that IRF4^f/f CD11c-cre-positive bone marrow chimeras had generally fewer colonic DCs compared to their IRF4^f/f cre-negative littermates, with DP DCs being the most affected subset. To test whether this decrease in DC numbers affected the uptake of parasite antigen in the colon, we injected 20 µg AF660-SEA in the colonic lamina propria. 24 hours after AF660-SEA injection, colons were harvested from injected IRF4^f/f CD11c-cre-positive and IRF4^f/f cre-negative bone marrow chimeras and digested with Collagenase V, Collagenase D, Dispase and DNase. Cells were stained for flow cytometry and levels of AF660 measured to indicate AF660-SEA uptake. We gated on total colonic DCs and observed that around 20% of cre+ and cre- cells had taken up AF660-SEA and that the total number of AF660-SEA⁺ colonic DCs was comparable between cre+

and cre- mice (Fig. 4-7 C&D). This again suggested that uptake of antigen was not affected in IRF4^f/f CD11c-cre-positive mice despite the fact that numbers of DCs were reduced in these mice. Taken together, we had observed no difference in the uptake of AF660-labeled SEA by small intestinal and colonic DCs in IRF4^f/f CD11c-cre-positive and IRF4^f/f cre-negative mice. This suggested that IRF4-deficiency and the decrease of DP DCs in the lamina propria of cre+ mice did not have an impact on antigen uptake and could not explain the impairment of Th2 responses in these mice.

4.1.5 CD11b-expressing dendritic cells are dramatically reduced